Project Summary T helper (Th) 17 cells are thought to play a key role in the development and pathogenesis of autoimmune diseases, including multiple sclerosis (MS) and its murine models, experimental autoimmune encephalomyelitis (EAE), rheumatoid arthritis, inflammatory bowel disease, and systemic lupus erythematosus. The development of Th17 cells is controlled by ROR?t, and small molecules that target ROR?t attenuate Th17 response and the severity of EAE and cutaneous inflammation. However, the regulation of ROR?t activity during Th17 cell differentiation by TCR signaling is largely unknown. Nedd4 (also known as Nedd4-1, neuronal precursor cell- expressed developmentally down-regulated 4) is a HECT-type E3 ubiquitin ligase. Through sequence analysis, we identified a PPLYKEL motif, an extended Nedd4 WW domain-binding motif, at the carboxyl terminus of the ROR?t ligand-binding domain, suggesting that ROR?t may be a binding partner of Nedd4. Indeed, Nedd4 is tyrosine-phosphorylated and activated upon TCR/CD28 stimulation and binds to ROR?t which undergoes K63- linked poly-ubiquitination. This ubiquitination is abrogated in T cells lacking Nedd4. Further analysis showed that although Nedd4 deficiency does not impair Th1, Th2, and inducible regulatory T cell differentiation, Th17 cell differentiation is greatly compromised in the absence of Nedd4. In support of this observation, mice deficient for Nedd4, or deficient for Nedd4 in T cells, have ameliorated EAE with impaired Th17 responses. Based upon these preliminary data, we hypothesize that upon T cell antigenic stimulation Nedd4 is activated by Src kinase(s), and targets ROR?t for K63-linked polyubiquitination, thus regulating Th17 responses and the susceptibility to EAE in mice and possibly MS in humans. In this proposal, we will investigate 1) How Nedd4 potentiates Th17 cell differentiation in vitro; 2) Whether Nedd4 promotes Th17 responses in vivo, regulating the susceptibility to EAE; and 3) How Nedd4 is activated in T cells in response to TCR/CD28 stimulation. The identification of Nedd4 as a key molecule regulating Th17 responses and the pathogenesis of EAE, and possibly MS, will have potential implications for using this molecule as a therapeutic target to treat autoimmune diseases involving Th17.